DataSheet_1_Distinct Bioenergetic Features of Human Invariant Natural Killer T Cells Enable Retained Functions in Nutrient-Deprived States.pdf

Invariant natural killer T (iNKT) cells comprise a unique subset of lymphocytes that are primed for activation and possess innate NK-like functional features. Currently, iNKT cell-based immunotherapies remain in early clinical stages, and little is known about the ability of these cells to survive and retain effector functions within the solid tumor microenvironment (TME) long-term. In conventional T cells (TCONV), cellular metabolism is linked to effector functions and their ability to adapt to the nutrient-poor TME. In contrast, the bioenergetic requirements of iNKT cells – particularly those of human iNKT cells – at baseline and upon stimulation are not well understood; neither is how these requirements affect effector functions such as production of cytokines and cytolytic proteins. We find that unlike TCONV, human iNKT cells are not dependent upon glucose or glutamine for these effector functions upon stimulation with anti-CD3 and anti-CD28. Additionally, transcriptional profiling revealed that stimulated human iNKT cells are less glycolytic than TCONV and display higher expression of fatty acid oxidation (FAO) and adenosine monophosphate-activated protein kinase (AMPK) pathway genes. Furthermore, stimulated iNKT cells displayed higher mitochondrial mass and membrane potential relative to TCONV. Real-time Seahorse metabolic flux analysis revealed that stimulated human iNKT cells utilize fatty acids as substrates for oxidation more than stimulated TCONV. Together, our data suggest that human iNKT cells possess different bioenergetic requirements from TCONV and display a more oxidative metabolic program relative to effector TCONV. Importantly, iNKT cell-based immunotherapeutic strategies could co-opt such unique features of iNKT cells to improve their efficacy and longevity of anti-tumor responses.

恒定自然杀伤T细胞（Invariant natural killer T, iNKT）是一类独特的淋巴细胞亚群，具备预激活特性并拥有先天NK样功能特征。当前，基于iNKT细胞的免疫疗法仍处于早期临床阶段，人们对这类细胞在实体肿瘤微环境（solid tumor microenvironment, TME）中长期存活并维持效应功能的机制知之甚少。在常规T细胞（conventional T cells, TCONV）中，细胞代谢与效应功能及其适应营养匮乏实体肿瘤微环境的能力紧密相关。与之相反，目前对iNKT细胞——尤其是人源iNKT细胞——在基线状态及受刺激后的生物能量需求尚不清楚，也不了解这些需求如何影响其细胞因子产生、细胞溶解蛋白表达等效应功能。本研究发现，与常规T细胞不同，人源iNKT细胞在接受抗CD3与抗CD28刺激后，其效应功能并不依赖葡萄糖或谷氨酰胺。此外，转录组分析显示，受刺激的人源iNKT细胞的糖酵解水平低于常规T细胞，且高表达脂肪酸氧化（fatty acid oxidation, FAO）与腺苷酸活化蛋白激酶（adenosine monophosphate-activated protein kinase, AMPK）通路相关基因。进一步观察发现，受刺激的iNKT细胞相较常规T细胞，拥有更高的线粒体质量与膜电位。实时海马代谢流分析结果表明，受刺激的人源iNKT细胞相较于受刺激的常规T细胞，更倾向于以脂肪酸作为氧化底物。综合上述数据，本研究表明人源iNKT细胞与常规T细胞具有不同的生物能量需求，且相较于效应型常规T细胞，其代谢程序更偏向氧化型。值得注意的是，基于iNKT细胞的免疫治疗策略可利用iNKT细胞的这一独特特性，以提升抗肿瘤应答的疗效与持久性。